title: Identification of protein partners of the human immunodeficiency virus 1 tat/rev exon 3 leads to the discovery of a new HIV-1 splicing regulator, protein hnRNP K.
abstract: HIV-1 pre-mRNA splicing depends upon 4 donor and 8 acceptor sites, which are used in combination to produce more than 40 different mRNAs. The acceptor site A7 plays an essential role for tat and rev mRNA production. The SLS2-A7 stem-loop structure containing site A7 was also proposed to modulate HIV-1 RNA export by the Rev protein. To further characterize nuclear factors involved in these processes, we purified RNP complexes formed by incubation of SLS2-A7 RNA transcripts in HeLa cell nuclear extracts by affinity chromatography and identified 33 associated proteins by nanoLC-MS/MS. By UV cross-linking, immunoselection and EMSA, we showed that, in addition to the well-known hnRNP A1 inhibitor of site A7, nucleolin, hnRNP H and hnRNP K interact directly with SLS2-A7 RNA. Nucleolin binds to a cluster of successive canonical NRE motifs in SLS2-A7 RNA, which is unique in HIV-1 RNA. Proteins hnRNP A1 and hnRNP K bind synergistically to SLS2-A7 RNA and both have a negative effect on site A7 activity. By the use of a plasmid expressing a truncated version of HIV-1 RNA, we showed a strong effect of the overexpression of hnRNP K in HeLa cells on HIV-1 alternative splicing. As a consequence, production of the Nef protein was strongly reduced. Interestingly also, many proteins identified in our proteomic analysis are known to modulate either the Rev activity or other mechanisms required for HIV-1 multiplication and several of them seem to be recruited by hnRNP K, suggesting that hnRNP K plays an important role for HIV-1 biology.
authors.affiliation: Laboratoire ARN-RNP Maturation-Structure-Fonction, Enzymologie Moléculaire et Structurale (AREMS), UMR7214 CNRS-UHP, Faculté des Sciences et Technologies, Nancy Université, Bld des Aiguillettes, Vandoeuvre-les-Nancy, France.
authors.forename: Virginie
authors.initials: V
authors.lastname: Marchand
authors.forename: Maryline
authors.initials: M
authors.lastname: Santerre
authors.forename: Christelle
authors.initials: C
authors.lastname: Aigueperse
authors.forename: Laetitia
authors.initials: L
authors.lastname: Fouillen
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Saliou
authors.forename: Alain
authors.initials: A
authors.lastname: Van Dorsselaer
authors.forename: Sarah
authors.initials: S
authors.lastname: Sanglier-Cianférani
authors.forename: Christiane
authors.initials: C
authors.lastname: Branlant
authors.forename: Yuri
authors.initials: Y
authors.lastname: Motorin
mesh_terms: D017398:Alternative Splicing; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D005091:Exons; D015967:Gene Expression Regulation, Viral; D015694:Gene Products, rev / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D034641:Heterogeneous-Nuclear Ribonucleoprotein K / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D010750:Phosphoproteins / Q000378:metabolism; D011485:Protein Binding; D012322:RNA Precursors / Q000235:genetics; D012326:RNA Splicing; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D016601:RNA-Binding Proteins / Q000378:metabolism; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000096:biosynthesis; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*; D000097796:Nucleolin
chemical_list: D015694:Gene Products, rev; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D010750:Phosphoproteins; D012322:RNA Precursors; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus; D000097796:Nucleolin